Two-dimensional acquisition for top-down native mass spectrometry of non-covalent protein complexes.

Informazioni relative al progetto

2D-TOPMASS

ID dell’accordo di sovvenzione: 101090276

DOI

10.3030/101090276

Progetto chiuso

Data della firma CE 27 Giugno 2022

Data di avvio 1 Luglio 2023

Data di completamento 30 Giugno 2025

Finanziato da

• Widening participation and spreading excellence

Costo totale

Nessun dato

Contributo UE

€ 166 278,72

Coordinato da

MIKROBIOLOGICKY USTAV AV CR V.V.I
Czechia

Periodic Reporting for period 1 - 2D-TOPMASS (Two-dimensional acquisition for top-down native mass spectrometry of non-covalent protein complexes.)

Periodo di rendicontazione: 2023-07-01 al 2025-06-30

Sintesi del contesto e degli obiettivi generali del progetto

Mass spectrometry (MS) is a powerful analytical technique able to describe the sequence, structure and modifications of a wide range of analytes from small molecules to large biomolecules such as proteins or nucleic acids. Since the invention of soft electrospray ionization (ESI) and namely in the last two decades, it has also found wide use in structural biology and biofarmaceutical industry to study higher order structure and interactions of large proteins and their assemblies up to intact viruses. This has been namely underpinned by the discovery of so-called "native mass spectrometry", which allows to transport proteins and their assemblies from solution into the gas phase (vacuum) inside the mass spectrometer gently enough to retain their non-covalent interactions, composition and often also their three-dimensional structure.

Recently, huge efforts have been aimed at expanding the boundaries of complex protein systems amenable to study by MS as well as at obtaining better and more detailed information about their composition and structural organisation. This is primarily hindered by two linked factors. First, by the difficulty analysing large native complexes, which require gentle handling and under native-enough solution conditions tend to not ionize well and handle only with difficulty in mass spectrometers, primarily due to the need of low acceleration energies as well as their low number of carried charges leading to very high mass-to-charge ratios, which are difficult to work with. And second - by the challenges to purposefully disrupt and break apart these complexes inside the mass spectrometer to obtain insights about their internal composition and organisation. This is because basic collisional fragmentation methods utilised in common mass spectrometers are often not powerful enough and provide only limited information. Hence, as these types of analyses are becoming increasingly vital for biopharmaceutical and medical applications, especially in so-called top-down approach, where proteins are analysed whole without prior enzymatic digestion to peptides, huge efforts are currently aimed at providing better fragmentation techniques. Namely in the form of analytical methods combining several techniques together which would yield more thorough sample characterization of challenging samples, which is sorely needed.

The project therefore aimed at:
1) designing and developing a suitable and practical source for native MS on an FTICR mass spectrometer
2) establishing native MS workflows for large proteins and protein / nucleic acid complexes on an FTICR MS
3) evaluating the performance of a range of fragmentation methods and their combinations for top-down MS
4) combining native top-down MS with two-dimensional acquisition schemes

Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti

Project 2D-TOPMASS aimed at utilising the power of a cutting-edge Fourier transform ion cyclotron resonance (FTICR) mass spectrometer towards the goal of better native top-down mass spectrometry and towards testing the use of advanced so-called two-dimensional ICR data acquisition schemes for non-covalent protein complexes.
In WP1, the project first tackled the lack of a suitable nano-electrospray platform for Bruker instruments, which would enable convenient and efficient manual native ESI analysis of more challenging samples. This first led to 3D printing and testing of a solution recently published by Götze et al. [JASMS 2023] and subsequently to a full-blown CAD designing and 3D print-supported manufacturing of an own solution. This resulted in a ESI source solution, which is robust, more precise to align and easier to handle with externally golden-coated glass capillaries commonly used for manual native MS. In WP2 the project tested several samples of increasing size on an FTICR and achieved the analysis of noncovalent protein complexes of increasing sizes up to about 150 kDa with variable mass resolution at a 15T Bruker solariX XR instrument. The project also endeavoured to identify easily available, reproducible and homogeneous testing samples in the (multi-)megadalton mass range, which will be followed up in follow-up work once these samples become available.

For top-down analysis, the mass spectrometer was previously custom-coupled with two powerful lasers able to irradiate ions inside the ICR detection cell in a precisely controlled fashion. In WP3 of this project, a custom-made dichroic mirror was installed in the optical path enabling parallel and/or sequential operation of the 193nm ArF excimer pulsed UV laser and the 10.600 nm CO2 continuous wave laser. Their operation individually as well as in different combinations and power settings were tested. For which custom pulse programmes (sets of instructions controlling the FTICR instrument's operation) were developed allowing diverse experimental arrangements. The results have shown that photodissociation with IR and UV laser benefits from their combined use, while there is also a dependence on the order of irradiation, where IR first followed by UV performed the best. On many samples there was also a marked difference observed between dissociating analyte ions inside an external collision cell when compared with mechanistically similar IR laser dissociation directly inside the ICR cell, where ions were then analysed and detected directly after the irradiation end. In WP4, dealing with two-dimensional acquisition of ions, protein top-down 2DMS experiments were tried with different setups. There, several obstacles were identified namely related to fragment ion signal intensities and signal-to-noise levels especially for UVPD. This led to the identification of better suited protein samples for follow-up projects. However, data acquired were also utilised in collaboration with colleagues in France to develop a novel approach to data processing in 2D acquisition yielding significantly improved ion metrics.

Tangentially, the project has led through the utilization of the techniques studied to several new collaborations on currently running or evaluated follow-up projects, which will further build on the results and methods developed in this project. These have also resulted in several publications published or in the final stages of preparation, where native MS has been used to describe and structurally characterize mutants in enzymes relevant for cancer and rare genetic disorders.

Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora)

The project has namely resulted in the following:

1) a robust and partially 3D printable nano ESI ion source compatible with Bruker instruments bearing the Apollo source, which were so far not available. This novel custom design is convenient for manually operated native MS and will be published and deposited in an open-source repository.

2) Hardware setup for combined UVPD / IRMPD and electron-based dissociation has been developed on a state-of-the-art FTICR platform and custom pulse sequences for instrument control were developed. This includes a custom hardware solution to enable UVPD dissociation in an LC-MS mode, which is not otherwise available on the instrument.

3) Combinations of photodissociation modes directly inside the ICR cell were shown to be beneficial for proteins on the FTICR platform. More research is currently ongoing in a follow-up project, which will expand the knowledge gained here as well as comparison with other instrumental platforms.

4) As a side product of this project, native MS helped uncover the previously unknown molecular mechanism disrupting the oligomerization of a GGPPS enzyme in multiple myeloma and present in important laboratory cell lines [Yehia et al. FEBS J. 2025], as well as deciphered the mechanism of human histone methyltransferase disfunction resulting in rare de novo genetic diseases in humans. [Hnizda et al., submitted, biorXiv: 10.1101/2025.09.25.678439v1]